PROJECT SUMMARY New technologies such as mass cytometry have greatly expanded our ability to deepen our understanding of the complexity of lymphocytes and related populations. The Stanford CCHI group has been particularly attuned to the potential in following the lead of the Nolan lab in exploiting this technology, with ground-breaking studies of T cells, NK cells, and cancer immunology. But a clear need has been for high dimensional methods to interrogate tissue sections in a wide variety of circumstances. This inspired several complementary efforts by Dr. Nolan and his group, specifically MIBI, which uses metal labeled antibodies in a high-resolution format, and CODEX (CODetection by inDEXing), a multi- parameter fluorescence-based imaging technology adaptable to most standard three-color fluorescence microscopes, and currently capable of sensitively and quantitatively measuring more than 60 markers in a single tissue. CODEX extends the deep phenotyping capabilities of multi-parameter flow cytometry while enabling the associated spatial context of a multitude of cell types, including rare cell types implicated in disease mechanisms. To achieve this high-parameter capability, antibodies against target epitopes are each tagged with unique DNA oligonucleotides and iterative cycles of imaging and removal of corresponding tags is performed to collect single cell proteomic measurements across all parameters. We will deploy CODEX for deep phenotyping of the 2D and 3D architecture of tonsil organoids. Recognizing a growing international biomedical and pharmaceutical interest in imaging applications to immunology, vaccine and drug development, this Technology Development Project will extend the current features of CODEX to deep phenotypic profiling of tonsil tissue architecture before and after exposure to influenza vaccine. Specifically, the Davis lab has developed a unique tonsil organoid system that can be exposed to a flu vaccine with subsequent production of high affinity antibodies several days to a week later. The versatility of this organoid system provides an unprecedented opportunity to modify and test influenza vaccine constructs and adjuvants in a fully human system and determine how best to trigger production of broadly neutralizing influenza antibodies, a goal toward generating a universal vaccine. We will extract feature data with this unprecedentedly deep data for the understanding of wholesale and minor tissue alterations that occur in response to influenza vaccine challenge?enabling a first ever map of ?tissue-omics? at the single cell level for the influenza vaccine response. We will also take advantage of the various manipulations that will be employed in Project 1 on this organoid system in order to gauge their effects on the organization of these cells and use this to formulate hypotheses regarding the significance of particular cellular groupings that we see in tonsils, which we refer to as ?neighborhoods?.